Copper, diammine[5,5'-bi-1H-tetrazolato(2-)-kN1, kN1']

Endpoint summary

Description of key information

According to these criteria, applying the CLP regulation N°1272/2008/EC and considering the ambiguous results of the present 442 B study, the tested substance CuDABT should be classified as sensitizer category 1B.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 may to 24 may 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

adopted 22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Provider : AUTOLIV ASP, 16700 W. Hwy 83, Promontory, UTAH 84307, USA
- batch No. P3656461
- Expiration date of the lot/batch: august 25th 2020
- Purity: 97.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a restricted area dedicated to explosive products, at outside temperature and humidity
- Solubility and stability of the test substance in the solvent/vehicle: not soluble in water

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no treatment

FORM AS APPLIED IN THE TEST (if different from that of starting material)
homogeneous suspensions in dimethyl formamide (DMF)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier LABS
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: 9 weeks
- Weight at study initiation: 20-25 g
- Housing: individually in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet ad libitum
- Water ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 10 per hour
- Photoperiod (hrs dark / hrs light): 12h/12h
- IN-LIFE DATES: From: To:11 May to 24 May 2016

Vehicle:

dimethylformamide

Concentration:

test item diluted in DMF at 50%, 25% and 10%

No. of animals per dose:

4 females

Details on study design:

- Compound solubility: test item is not soluble in water or other vehicle tested. However, a homogeneous blue suspension was obtained with 10% and 50% of dimethyl formamide (DMF). 25% of DMF was chosen as vehicle.

PRELIMINARY SCREENING TEST
preliminary test was performed using one mouse treated daily (days 1,2 and 3) with a diluted test item at 50% in DMF
no mortality and no signs of systemic toxicity were noted
no cutaneous reaction (erythema) was noted at the tested concentration of 50%
the percent in ear thickness increase was 9.1% on day 3 versus day 1 and 14.3% on day 6 versus day 1
therefore, 50% was chosen as the highest concentration for the main test

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
3 groups of female mice were treated for 3 consecutive days with 50 µL (25µL per ear) of the test item diluted at concentrations of 50%, 25% and 10% in dimethylformamide (DMF). A vehicle group of 4 mice was treated with DMF. On day 5, 0.5mL of BrdU solution (10 mg/mL) mas injected by the intraperitoneal route. On day 6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocytes using an ELISA kit.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

no statistics

Positive control results:

BrdU index (mean): 1.630, stimulation index SI: 1.92 +/- 0.36.

Key result

Parameter:

SI

Value:

2.44

Variability:

0.43

Test group / Remarks:

test item 10%

Key result

Parameter:

SI

Value:

1.88

Variability:

0.26

Test group / Remarks:

test item 25%

Key result

Parameter:

SI

Value:

1.62

Variability:

0.1

Test group / Remarks:

test item 50%

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION: according to O.E.C.D. guideline No.442-B, the result obtained here is borderline positive results as Stimulation Index (SI) values between 1.6 and 1.9 have been obtained (considering the average and deviation).
The maximum SI value, registered at the lowest dose, may be explained by a best bioavailability of the test item. Indeed at all concentrations, the preparations were a blue suspensions and despite a vigorous homogenisation before each treatment, a sedimentation was noted which confirm that tested concentration are aboce the solubility limit. This sedimentation was higher in the group treated at the highest concentration of 50%.

EC1.6 CALCULATION: can not be determined as all SI values were higher than 1.6 (borderline positive)

CLINICAL OBSERVATIONS: no mortality and no signs of systemic toxicity, no erythmae were noted in the test and control animal during the test

BODY WEIGHTS: bodyweight changes of the test animals between day 1 and day 6 were comparable to those observed in the corresponding control group animals over the same period and are not consodered as relevant.

Local irritation: test item has to be considered as not excessively irritant at these concentrations

test item	ear weight increase on day 6	ear thickness increase on day 3 versus day1	ear thickness increase on day 6 versus day 1	cutaneous reaction
10%	7.5%	5.0 +/-5.8	-3.4 +/-7.1	no
25%	7.3%	3.6 +/-4.6	-2.4 +/-6.1	no
50%	5.5%	7.5 +/-3.2	5.3 +/-10.8	no

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

According to the criteria, applying the CLP regulation N°1272/2008/EC and considering the ambiguous results of the present 442 B study, the tested substance CuDABT should be classified as sensitizer category 1B (also see Sensitisation Summary for more details). The signal word "warning" and the hazard statemnt H317 "may cause an allergic skin reaction" are required.

Executive summary:

The test item was performed to assess the skin sensitisation potential of the test item Copper Dianmmine Bitetrazole (CuDABT) in the CBA/J strain mouse following topical applications to the dorsal surface of the ear. The basic principle underlying the LLNA:BrdU is that sensitizers induce proliferation of lymphocytes in the lymph nodes drainning the site of the test item application. The experimental protocol was established according to the O.E.C.D. Guideline No.442 -B adopted 22 July 2010.

3 groups of female mice were treated for 3 consecutive days with 50 µL (25µL per ear) of the test item diluted at concentrations of 50%, 25% and 10% in dimethylformamide (DMF). A vehicle group of 4 mice was treated with DMF. On day 5, 0.5mL of BrdU solution (10 mg/mL) mas injected by the intraperitoneal route. On day 6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocytes using an ELISA kit.

No mortality and no signs of systemic toxicity were noted in the test and control animals during the test.

The percent in ear thickness increase was -3.4 +/-7.1, -2.4 +/-6.1, 5.3 +/-10.8 on day 6 versus day 1, in mice treated with test item at 10%, 25% and 50%. The percent in ear weight increase was 7.5%, 7.3%, 5.5% on day 6 in mice treated with test item at 10%, 25%, 50%. No cutaneous (erythema) was noted. The test item has to be considered as not excessively irritant at these concentrations in accordance with the O.E.C.D. criteria.

The Stimulation Index (SI) calculated by individual approach was 2.44 +/-0.43, 1.88 +/-0.26, 1.62 +/-0.10 for treated groups at 10%, 25%, 50%. which is ambiguous according to the 442B OEDC guideline. The maximum SI value, registered at the lowest dose, may be explained by a best bioavailability of the test item. Indeed at all concentrations, the preparations were a blue suspensions and despite a vigorous homogenisation before each treatment, a sedimentation was noted, which means that the tested solution was above the solubility limit. This sedimentation was higher in the group treated at the highest concentration of 50%.

The results obtained in these experimental conditions, enable to conclude that the test item Copper Diammine Bitetrazole (CuDABT) has to be classified as a sensitiser in Category 1B, in accordance with the Regulation EC No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to guidance R7a. and by definition, a skin sensitiser is an agent that will lead to an allergic response in susceptible individuals following skin contact. As a consequence of a secondary - usually organ-specific - subsequent re-exposure, adverse health effects on the skin (allergic contact dermatitis).

An OECD 442B test has been conducted according to annex VII to assess any sensitizer properties of CuDABT.

No mortality and no signs of systemic toxicity were noted in both the CuDABT and control animals during the test. No cutaneous erythema was noted. The Stimulation Index (SI) calculated by individual approach was 2.44 +/-0.43, 1.88 +/-0.26, 1.62 +/-0.10 for treated groups at 10%, 25% , 50%. The EC 1.6 could not be determined as all SI values were higher than 1.6.

As per OECD guidance: “For a borderline positive response between an SI of 1.6 and 1.9, (which is the case here), users may want to consider additional information such as dose-response relationship, evidence of systemic toxicity or excessive irritation, and where appropriate, statistical significance together with SI values to confirm that such results are positive (10). Consideration should also be given to various properties of the test substance, including whether it has a structural relationship to known skin sensitizers, whether it causes excessive skin irritation in the mouse, and the nature of the dose-response observed. These and other considerations are discussed in detail elsewhere”

In this present study the dose-response is not usable, in part because of the very poor solubility of the test substance. There is no dose-response curve which confirms the dose-effect relationship in the present study.

Considering that the low solubility of the substance may interfere with the accuracy of the test, other parameters have to be considered to confirm if the borderline result should be considered as positive or negative:

• Evidence of systemic toxicity: there is no evidence of systemic toxicity

• Excessive irritation: there are no signs of irritation in the study

OECD also states: “Collecting data at the level of the individual mouse will enable a statistical analysis for presence and degree of dose-response relationship in the data. Any statistical assessment could include an evaluation of the dose-response relationship as well as suitably adjusted comparisons of test groups (e.g. pair-wise dosed group versus concurrent solvent/vehicle control comparisons).”

There is no data in the study that justifies the dose-response relationship and as a consequence no dose-response based findings are available to justify a positive response.

According to the Guidance R7a., substances shall be classified as skin sensitisers (Category 1) where data are not sufficient for sub-categorisation in accordance with the following criteria:

(a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons; or

(b) if there are positive results from an appropriate animal test (see specific criteria in paragraph 3.4.2.2.4.1).

According to these criteria, applying the CLP regulation N°1272/2008/EC and considering the ambiguous results of the present 442 B study, the tested substance CuDABT should be classified as sensitizer category 1B.